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Molecular roots of FGFR3-related skeletal dysplasia

Molecular roots of FGFR3-related skeletal dysplasia. Pavel Krejci , PhD Institute of Experimental Biology, Masaryk University, Brno Department of Cytokinetics, Institute of Biophysics AVCR, Brno Cedars-Sinai Medical Center, Los Angeles, USA.

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Molecular roots of FGFR3-related skeletal dysplasia

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  1. Molecular roots of FGFR3-related skeletal dysplasia Pavel Krejci, PhD Institute of Experimental Biology, Masaryk University, Brno Department of Cytokinetics, Institute of Biophysics AVCR, Brno Cedars-Sinai Medical Center, Los Angeles, USA

  2. NavaznostiPrednaska navazuje na obecne vymezeni podstaty bunecneho signalovaniObecne zakonitosti budou demonstrovany na priklade FGF signalingu v skeletalni dysplasii

  3. How do the limbs grow? age resting cartilage proliferating cartilage bone proliferating cartilage resting cartilage

  4. FGFR3-related skeletal dysplasia Achondroplasia

  5. FGFR3-related skeletal dysplasia STATURE I AC Hypochondroplasia Achondroplasia SADDAN Thanatophoric Dysplasia II FGF binds here III TM TK

  6. Thanatophoric Dysplasia healthy - short long bones - brachydactyly - macrocephaly - low nasal bridge - spinal stenosis - temporal lobe malformations TD

  7. resting proliferating hypertrophic bone healthy TD

  8. Sahni et al., Genes Dev 1999, 13, 1361-66. Sahni et al., Development 2001, 128, 2119-29. FGFR3 ? STAT1 ? CKI ? FGFR3-related skeletal dysplasia Growth arrest

  9. FGF inhibitschondrocyte proliferation by arresting their cell cycle in G1 phase 3H-thymidine (cpm x 103) % of cells FGF2 (h) FGF concentration (ng/ml)

  10. ….via inhibition of cdk activity necessary for progression through the G1 phase of a cell cycle Krejci et al., Exp Cell Res 2004, 295, 152-64

  11. FGF alters the cartilage-like phenotype of chondrocytes FGF (h) control FGF - 0.5 1 4 8 12 24 48 72 aggrecan collagen II collagen I GAPDH

  12. ……via MMP-mediated degradation of extracellular matrix control FGF2 control FGF2 MMP2 proMMP9 matureMMP9 MMP3 proMMP2 proMMP2 MMP9 intermediate MMP10 matureMMP2 MMP13 pro MMP3/10 mature MMP3/10 GAPDH proMMP13 matureMMP13 Krejci et al., J Cell Sci 2005, 118, 5089-100

  13. FGF2 activates Erk and p38 MAPK, PLCg and PKB in chondrocytes FGF2 C1 C2 1’ 5’ 10’ 30’ 1h 2h 4h 8h P-Erk1/2 Erk1/2 P-p38 p38 P-PLCg1 PLCg1 1’ 5’ 30’ 1h 4h 6h 12h 18h 24h 0h 4h 12h F F/H F F/H F F/H F F/H F F/H F F/H F F/H F F/H F F/H- - H - H P-PKB PKB

  14. ……but only Ras/Erk activity is involved in FGF-induced growth arrest(Krejci et al., Exp Cell Res 2004, 295, 152-64) SU5402 80 Ras RasN17 60 40 cells/wellx103 cell colonies/100 cells (%) 20 0 - F1 F10 F100 U0126 80 RasV12 60 40 cells/wellx103 cells/wellx103 20 0 .001 .01 .1 1 10 100 Inhibitor concentration (mM) - U0126

  15. Erk MAP kinase activity is necessary for FGFR3 phenotype in cartilage Murakami et al., Genes Dev 2004, 18, 290-305. Raucci et al., J Biol Chem 2004, 279, 1747-1756. Krejci et al., Exp Cell Res 2004, 297, 152-164. Murakami et al., Genes Dev 2004, 18, 290-305. Raucci et al.,J Biol Chem 2004, 279, 1747-1756. Krejci et al., Exp Cell Res 2004, 297, 152-164.

  16. C-type Natriuretic Peptide (CNP) rescues achondroplastic phenotype in FGFR3-ACH mice. Yasoda et al., Nature Medicine 2004, 10, 80-86

  17. CNP counteracts FGF2-mediated chondrocyte growth arrest through cGMP-dependent pathway 50 control 40 30 cells per well [x104] control 20 FGF2 10 FGF2 0 _ 0.1 0.2 0.5 1_ _ _ _CNP [mM] _ _ _ _ _ 10 100 200 500pCPT-cGMP [mM]

  18. CNP antagonizes FGF2-mediated loss of cartilage extracellular matrix in chondrocytes unstimulated FGF2 control CNP pCPT-cGMP

  19. CNP counteracts FGF2-mediated activation of Erk MAP kinase in chondrocytes FGF2 _+ ++ + _ _ + + + + _FGF2 _ _0.1 0.2 0.5 0.1_ _ _ _ _ _CNP [mM] __ _ _ _ _ _ _100 200 500 100pCPT-cGMP [mM] FGFR3 P-Erk1/2 FRS2 Erk1/2 Ras FGF2_ + + _ CNP_ _ ++_Ab FGF2_ ++ _ CNP_ _ ++ IP:Raf-1 Raf-1 Raf-1 Ras-GST WB P-MEK MEK Ras total P-Erk1/2 P-Erk1/2 Erk Erk1/2 Erk1/2

  20. CNP inhibits Erk MAP kinase module at the Raf level FGF2 CNP FGFR3 NP-R FRS2 cGMP STOP Ras PKG Raf-1 Growth arrest MEK Erk Matrix degradation Krejci et al., J Cell Sci 2005, 118, 5089-100

  21. Is protein kinase C (PKC) involved in FGFR3- mediated activation of Erk in chondrocytes? FGF2 FGF2_+ ++ + _ ++ + __ + ++ + _ Gö6983 (mM)__ 1 5 10 5 _____ _ _ _ _ _ Bis I (mM)______ 1 5 10 5 _ _ _ _ _ _ Gö6976 (mM)____________ 1 5 10 5 FGFR3 FRS2 Ras P-Erk1/2 Raf-1 Erk2 MEK FGF2_+ ++ + + ++ + _ GFX (mM)__ 0.5 1 5 10 20 50 _ 10 vehicle (DMSO)__ + ++ + + ++ + kinase Erk P-Elk

  22. FGF2_+ + _ Bis I__ + + FGF2_+ + _ Bis I__ + + IP:Raf-1 FGF2 Raf-1 WB Ras-GST FGFR3 MEK1 Ras total FRS2 -MEK1 -MEK1 -MEK1 P P P MEK1 Ras FGF2 _ ++ ++ ++ +++ _ _ Bis I(mM)_ _ 0.5 1 5 10 20 50 _ _ 10 _ Raf inhibitor(mM)____ _ _ __ 1_ _ 1 vehicle (DMSO)__++ + + + + + + + + Raf-1 kinase MEK Raf-1 Erk MEK1

  23. FGF2_ ++ ++ ++ +++ + Bis I(mM)__ 1 5 10 20 50_ _ _ _ SU5402(mM)_ _ _ _ _ _ _1 10 20_ vehicle(DMSO)__ + + + + + + + + + FGF2 kinase FGFR3 FRS2 Gab1 IP:FRS2 SHP2 SHP2 GRB2 SOS WB FRS2 -Y-FRS2 P FGF2_5’ 10’ 30’ 1h_30’ 30’_ Bis I_ __ __ _ _+ + IP:Gab1 SHP2 GRB2 SOS WB Ras Gab1 FGFR3 Raf-1 FRS2 MEK Erk

  24. Bis I Bis I Shp2-Grb2-SOS Protein kinase C inhibitor Bisindolylmaleimide I (Bis I)suppresses the FGF2-mediated activation of Erk MAP kinase pathway in chondrocytes by preventing the SHP2 association with FRS2 and Gab1 adaptor proteins FGF2 FGFR3 ? FRS2 Grb2-SOS Gab1 SHC Ras Raf-1 MEK Erk Krejci et al., J Biol Chem 2007, 282, 2929-36

  25. FGFR3-K508M FGFR3-wt FGFR3-K650M GFP FLAG actin IP: FLAG FLAG WB STAT1 FGFR3-K650E FGFR3-wt substrate: STAT1++ ++ ++ SU5402(mM)_ +_ _ +_ ATP++_++_ FGFR3 associates with STAT1 and acts as STAT1-kinase in chondrocytes FGFR3 ? STAT1 ? ? CKI P-Y701-STAT1 Growth arrest STAT1 FGFR3

  26. STAT1 and STAT3 are not involved in FGFR3-mediated growth arrest in chondrocytes FGFR3 STAT1 ? ? CKI Growth arrest Krejci et al., J Cell Sci. In revision

  27. 150kDa STAT3-YFP 100 STAT3 75 STAT3-YFP DAPI merge DIC/merge control FGF2 2h FGF2 48h IL6 30m FGF2 48h IL6 30m Chronic FGF stimulus inhibits cytokine/STAT signaling in chondrocytes

  28. FGF2 (72h)_ +_+_ +_ +_+ IL6__++______ IL11____++__ _ _ LIF______ ++_ _ IFNg________++ FGF2 (48h)_ +_+_ +_ +_+ IL6 (minutes) __5 5 10 10 30 30 60 60 P-Y705-STAT3 1.2 STAT3 0.9 actin STAT3 activation (OD450-650nm) 0.6 0.3 0 Chronic FGF stimulus inhibits cytokine/STAT signaling in chondrocytes

  29. FGF2 IL6 IL11, LIF FGFR3 IL6RA Shp2 Frs2 gp130 Cish Socs1 Socs3 STAT3 nucleus STAT3 Chronic FGF stimulus inhibits cytokine/STAT signaling in chondrocytes Krejci et al., manuscript in preparation

  30. control FGF2 FGF2 [ng/ml] - 5 10 20 48 36 24 12 0 SA-b-gal FGF2 causes premature senescence in chondrocytes SA-b-gal positive cells per 50 cells [n=10] Krejci et al., manuscript in preparation

  31. FGFR3 recruits multiple adapter proteins to activate Ras/Erk signaling pathway Krejci et al., manuscript in preparation

  32. FGF2 signals towards the cytoskeleton in chondrocytes Phalloidin DAPI merge control FGF2

  33. FGF2 C1 15‘ 1h 3h 6h 12h 24h 48h 72h C2 C3 b-catenin actin FGF2 accumulates b-catenin in chondrocytes

  34. 20012007 FGFR3 IL6, LIF, IL11, IFNg FGFR3 CNP ? PKC NPR-B STAT1 Frs2, Gab1, SHC STAT1/3 STAT1 ? cGMP CKI ? Ras/Raf/MEK/Erk PKG Growth arrest CKIMMP Growth arrest Matrix degradation

  35. From bench to bedside: Strategies to treat achondroplasia 1. Stable CNP analog – Biomarin Pharmaceutical Inc. 2. Neutralizing antibody to FGFR3 3. Small chemical inhibitor of FGFR3 Prochon Biotech Ltd.

  36. UCLA, Los Angeles California Robert Pogue Matthew Schibler Laboratory of Molecular Embryology MZLU Brno, Czech republic Vita Bryja Jiri Pachernik INSERM U589, Toulouse, France Herve Prats Bernard Masri Vincent Fontaine • Cedars-Sinai Medical Center • Los Angeles, California • William Wilcox • Katerina Pejchalova • Betty Mekikian • Patricia Lin • Matthew Rock • Claire Rock • UCI, Irvine • California • Leslie Thompson • Tamara Kashiwada • Lisa Salazar

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